Bladder cancer (BC) is the fourth most common cancer in men with more than 70,000 new cases and 14,680 deaths in 2010. Major risk factors of BC include tobacco smoking and occupational exposure to aromatic amines in the dye and rubber industries. These exposures are thought to account for about 50% of bladder cases. Several epidemiological studies have linked the consumption of well-done cooked meat containing structurally related heterocyclic aromatic amines (HAAs) with BC risk. Endogenous lipid peroxidation products also may contribute to bladder mutagenesis. However, the specific chemicals that contribute to BC are largely unknown. Urine is a promising biospecimen for the analysis of cancer biomarkers. For example, vesicles (exosomes) excreted in urine serve as carriers of genetic information and a novel source of cancer biomarkers. We propose that exfoliated urinary cells also may serve as a non-invasive biospecimen to assess DNA damage of the upper and lower urinary tract. The goal of this grant renewal, New biomonitoring methodologies to measure DNA adducts in human tissues is to validate exfoliated urinary cells as a source of specimen to assess exposures to chemicals, some of which may contribute to upper urothelial cancer (UUC) and BC. We will examine DNA adducts formed in exfoliated urinary cells and in urothelial tissues from exposure to environmental, dietary, medicinal, and endogenous genotoxicants. Our preliminary data demonstrate that DNA adducts of aristolochic acid (AA), a urothelial carcinogen found in traditional Chinese medicine (TCM) or as a contaminant of flour, can be quantified in exfoliated urinary cells, by ultraperformance liquid chromatography/multistage scan mass spectrometry (UPLC/MSn), a method with superior sensitivity to that of the commonly used but non-specific 32P-postlabeling assay. We propose to use exfoliated urinary cells as a noninvasive source of biospecimen to assess DNA damage, using UPLC/MSn, in the upper urothelium and/or bladder of subjects at risk of developing UUC and BC. In Aim 1, we examine DNA adducts of AA in urothelial tissues and exfoliated urothelial cells of subjects exposed to AA via TCM or contaminated bread. In Aim 2, we establish techniques to measure DNA adducts of other genotoxicants, that can damage bladder, including aromatic amines, HAAs, and lipid peroxides in the urothelium and urinary cells of mice exposed to these chemicals or to tobacco smoke, using targeted and adductomics scanning approaches. In Aim 3, MS methods are applied to measure DNA adducts formed in urothelium and exfoliated urinary cells of patients who have undergone cystectomy for BC, and urinary cells of non-smokers and smokers. Our proposed research is highly relevant to the NIEHS mission, which is to understand how environmental exposures contribute to health outcomes. Exfoliated urinary cells are an untapped biospecimen that can be used to screen for damage to DNA induced by chemicals that contribute to the etiology of BC.